Pristimerin inhibits the proteosome and NF-κB activity. (A,B) The direct effect of pristimerin on the chymotrypsin-like peptidase activity of purified human 20S proteosome enzyme was assayed using a fluorogenic peptide substrate, suc-LLVY-AMC, and compared with proteosome chymotrypsin-like activity in the absence of drug or in the presence of bortezomib. (A) Time course of 20S proteosome cleavage of Suc-LLVY-AMC, in the presence (□) or absence (■) of drug, at specified concentrations, monitored by release of free AMC fluorophore (measured in arbitrary fluorescence units, AFU) at 2-minute intervals. Each square represents the mean result from duplicate wells. Linear accumulation of AMC was observed during the first hour. (B) Purified 20S proteosome chymotrypsin-like activity, calculated as the velocity of AMC release (in units of AFU/min), and dose-response curve for pristimerin (□) demonstrating an IC₅₀ for inhibition of chymotrypsin-like activity below 30 to 60 nM. Data represent the mean velocity ± SEM per concentration. (C,D) To assess the effect of pristimerin on intracellular proteosome and NF-κB function, H929 myeloma cells were treated with pristimerin for 2 hours at various concentrations (C), or with pristimerin 500 nM for various durations (D). Lysates (10 μg per lane) were immunoblotted for ubiquitinated protein (top panel), α-tubulin (loading control: middle panel) or for IκB (D bottom panel); pristimerin causes accumulation of ubiquitinated high-molecular-weight protein, consistent with proteosome inhibition, and parallel suppression of phosphorylated IκB, consistent with inhibition of IKK. (E) To confirm parallel inhibition of IKK by pristimerin, H929 myeloma cells were treated as specified for 3 hours and assessed by immunoblot for inhibition of phosphoactivation of IKKα and IKKβ (using a S180/S181 phosphorylation-specific antibody) and for downstream phosphorylation of IκB. (F,G) Trans-activating activities of NF-κB, AP-1, TCF4, or CRE transcription factors in (F) U293FT kidney cells and (G) KMS11 myeloma cells, after exposure to pristimerin 500 nM or DMSO vehicle for 6 hours. Cells were transfected with transcription factor–specific luciferase reporter plasmids 30 hours prior to drug treatment. Relative to the other transcription factors, NF-κB trans-activating activity was greater in KMS11 myeloma cells than in 293 cells, consistent with biallelic loss of the NF-κB–negative regulator TRAF3 in KMS11.²⁶  Allowing for a luciferase half-life of approximately 3 hours, NF-κB activity was profoundly suppressed in KMS11 by pristimerin (4-fold suppression of luciferase is observed in 2 half-lives). Positive and negative controls for luciferase detection were 2 irrelevant lysates containing, or lacking, luciferase. Data are represented as the mean of duplicate samples ± range.